The heart is the first organ to develop, a process that involves multiple cell fate decisions and morphological processes. Key among these is the orientation of specific morphological events such as trabeculation and septation toward the heart lumen. Failure of these processes can result in congenital heart disease, a major cause of mortality and morbidity. The objective of this proposal is to identify the mechanism whereby atypical Protein Kinase C Iota (Prkci) and the downstream PAR polarity machinery direct myocardial polarization at the single cell and whole organ levels. The central hypothesis is that directional cues from the endocardium and cardiac jelly direct Prkci and the Par complex to orient the spindle apparatus and the cell division plane of luminal myocardial toward the heart lumen and propel trabecular formation. This hypothesis has been formulated on the basis of strong preliminary data produced in the applicant's laboratory and is tested with three specific aims: 1) determine how Prkci directs cardiomyocyte proliferation and differentiation during ventricular trabeculation; 2) define the molecular mechanism through which Prkci controls the Par complex and the spindle machinery to regulate polarized cell division in luminal myocardial cells; and 3) define the non-cell autonomous inductive signaling cues that direct myocardial polarization. Under the first aim, single cell clonal analysis is performed to determine how Prkci directs oriented cell division. Highly innovative cell labeling and genome editing techniques are coupled with cutting-edge spectral confocal microscopy to define the role of Prkci-dependent polarized cell division in the initiation of trabeculation. In the second aim, the applicant determines how Prkci controls the downstream Par machinery and explores the potential of this pathway to direct polarized cell division in human stem cell derived myocytes. In the third aim, the applicant identifies the upstream inductive cues that feed into Prkci and its interacting partners and adapts the knowledge gained from developmental biology to stem cell biology. The rationale for these studies is that they are the first to address how the highly conserved PAR complex orients the mitotic spindle and axis of cell division to control myocardial alignment during in vivo cardiogenesis and in vitro cellular differentiation. The advent of stem cell based approaches to regenerative cardiology and the necessity of ensuring the proper cellular alignment of transplanted cells or constructs make this research highly significant. Thus, the overall impact of this project is to provide a mechanistic understanding of how the highly conserved Par machinery directs polarized cell division to control normal cardiac organogenesis. This study provides a fundamental understanding of a key morphological event in cardiac development that can be applied to regenerative cardiovascular medicine where it is necessary to expand the number of functional CMs and promote their alignment and cellular integration with the native myocardium.